The present invention relates generally to the assay of biological specimens. More particularly, it relates to improved compositions and methods for enzyme immunoassays.
An antibody is a protein synthesized by an animal in response to the presence of a foreign substance or antigen. Antibodies, also known as immunoglobulins, have specific affinity for the antigens that elicit their synthesis. Because of the high degree of specificity that antibodies exhibit, the antigen-antibody interaction in vitro is widely used for diagnostic purposes.
Immunoassays (IA), for example, use this specificity to measure the presence of antigens or antibodies in biological samples. Referring to FIGS. 1A-C, the technique is illustrated for a simple monovalent antigen. In FIG. 1A, a membrane having a known receptor (e.g., antibody (Ab)) specific for the molecule of interest is provided. In FIG. 1B, a sample of analyte has been placed on the membrane, and after allowing a sufficient period of time, antigen-antibody complexes (Ag-Ab) form. Any free antigen remaining is separated from the membrane by conventional methods. The amount of antibody sites occupied by antigen is proportional to the amount of antigen present in the sample.
In FIG. 1C, "labeled" antibody (Ab*) is added. "Labeled" antibody, which is conjugated with a moiety that may be easily detected, competes for the same epitope. After allowing sufficient time for the formation of complexes (Ab*-Ag), unbound antibody is removed by conventional methods. The amount of labelled molecules remaining after separation indicates the amount of reaction, and hence the presence (or absence) of a particular analyte. Numerous other assay formats are known and reported in the medical and patent literature.
Several different labels have been employed in IA. Radioimmunoassay (RIA), for example, uses radioactive isotopes as labels. Reaction is detected by the presence of radioactivity.
Enzyme immunoassay techniques, e.g., enzyme-linked immunosorbent assay (ELISA), use antibody or antigen that is conjugated or labelled with an enzyme, for example, alkaline phosphatase. As shown in FIG. 1D, detection of an enzyme-labeled antibody is achieved by the addition of a substrate to the reaction. The enzyme can rapidly convert an added colorless substrate into a color product (e.g., hydrolysis of indoxyl phosphate), or a non-phosphorescent substrate into an intensely fluorescent product. Appearance of a characteristic color that results from the activity of the enzyme on the substrate signifies the presence or absence of analyte.
Because they are less expensive, enzyme immunoassays are rapidly replacing RIA techniques. In addition, special precautions for handling radioactive material are not required. Nevertheless, enzyme immunoassay techniques maintain the high degree of specificity common to immunoassays.
Of particular interest to the present invention is a class of assays referred to as membrane assays. Such assays usually employ a capture antibody or antigen immobilized on a permeable membrane. A sample is applied to and drawn through the membrane, typically by an absorptive layer in contact with the backside of the membrane. The analyte of interest is captured and can be detected by the subsequent application of an enzyme-labeled antibody specific for the analyte.
In large part, immunoassays have been utilized in clinical laboratories to quantitatively determine a wide variety of compounds, particularly drugs and biological markers, such as hormones. Immunoassays have also found less expensive application where semi-quantitative or qualitative results are acceptable, particularly where the determination involves untrained personnel. Even in clinical laboratories though, simple and rapid screening tests shorten lab turnaround time and lower operating costs.
In developing an immunoassay, particular features are desirable. These include, for example, the ease of preparation of the reagents, the accuracy with which samples and reagent solutions must be measured, and the storage stability of the reagents. In developing an assay which could have application with untrained personnel, such as a test to be performed in the home, the observed result should be minimally affected by variations in the reagents used.
Prior techniques, however, employ reagents which are not known for their stability, i.e., the reagents are prone to spontaneous denaturation. For example, the enzyme currently employed in many ELISA and membrane systems, calf intestine alkaline phosphatase (CIAP), is relatively heat labile. Unless it is lyophilized, it is unstable. As a result, it is not available in a convenient form, such as a liquid. The lack of stability of current reagents not only decreases convenience and reliability but also adds to the ultimate cost of the assay as well.
In enzyme immunoassays, it is desirable to have stable reagents which are available in a convenient form. Preferably, the reagents should be economical to produce and be available as a liquid. However, an immunoassay should not be compromised by a reagent which lacks sufficient activity for detection. Thus, it is desirable to employ stable reagents which can be detected, either readily or with enhancement. The present invention fulfills these and other needs.